The objective of this R29 proposal is to understand what role inactivation of the p16 tumor suppressor gene plays in the multi-stage development of oral cancers, with an emphasis on premalignant lesions. The studies described herein address the hypothesis that a subset of the genetic changes commonly present in end-stage oral squamous cell carcinomas (SCC) will have already occurred in premalignant (leukoplakia, erythroplakia and proliferative verrucous leukoplakia) lesions. The p16 gene has previously been shown to be inactivated through a variety of mechanisms in 80 percent of SCC of the head and neck. In contrast, a critical evaluation of the role of p16 inactivation in premalignant oral lesions has yet to be explored. Therefore, we will focus our studies on this critical cell cycle regulatory gene in premalignant cells. The experimental approach involves a combination of genetic and biochemical methodologies. Studies in Specific Aim 1 are designed to determine the incidence and mechanism of p16 inactivation (including deletions, insertions, single nucleotide substitutions, and gene hypermethylation events) in premalignant oral lesions. Secondary to our evaluation of premalignant lesions, SCC will be screened for p16 mutations in an effort to identify a larger number of missense mutations for subsequent studies in Specific Aims 2 and 3. Once identified, premalignant and malignant oral lesions exhibiting p16 mutations will be assessed at the level p16 transcription and translation for altered p16 expression compared to patient-matched normal tissues (Specific Aim 2). These expression studies will be performed as a means of determining a precise level of p16 deregulation. In addition, specific P16 mutant proteins will be evaluated in Specific Aim 3 for their degree of biologic activity compared to the wild-type P16 protein. Mutant P16 proteins will be constructed by site-directed mutagenesis and evaluated for their ability to bind to CDK4 and inhibit CDK4 kinase activity in vitro. The in vivo functional activity of specific mutant P16 proteins will also be evaluated indirectly by determining the levels of phosphorylated and unphosphorylated RB proteins in samples derived from individual premalignant and malignant oral lesions. Overall, it is anticipated that the results from these studies will not only broaden our understanding of the molecular mechanisms of oral cancer development, but may also aid in the identification of specific causative agents, intermediate endpoint biomarkers, and the development of successful intervention strategies which target premalignant disease.